Analysis of the molecular weight of nucleic acids is typically performed using a technique called agarose gel electrophoresis (C. F. Simpson, ELECTROPHORETIC TECHNIQUES, Academic Press, New York (1983)). Alternatively, polyacrylamide can be used instead of agarose. The process of agarose gel electrophoresis involves the formation of a solidified agarose slab gel with pre-formed wells on one end for holding solutions containing nucleic acids. The agarose gel is placed into a box with electrodes on each end and submerged in a buffer that allows for the conduction of an electrical current through the agarose which facilitates the migration of the nucleic acids through the matrix. The electrical field applied results in the negatively charged nucleic acid molecules migrating towards the positively charged pole. The movement of nucleic acids through the porous agarose matrix results in the separation of both DNA and RNA based on molecular weight.
In order for a nucleic acid to be resolved in agarose, it must be applied to the well prior to applying the electric current. Because the agarose gel is submerged under a buffer, it is important to hold the nucleic acid in place within the well of the gel. This requires that the nucleic acid must first be mixed with a solution which contains a compound that adds weight to the sample, thereby allowing the nucleic acid to sink to the bottom of the well and avoid diffusion into the buffer. Weight may be provided from a number of suitable high-density compounds including sucrose, trehalose, glycerol, sorbitol, Ficoll™, and other sugars of various molecular weights.
The typical compound used in loading formulations is Ficoll™ because its high density allows for less diffusion of the nucleic acid in the well, thus providing sharper bands in the agarose gel. Ficoll™ also offers the advantage of working well in the two common gel buffers: Tris-Acetate-EDTA (TAE) and Tris Borate-EDTA (TBE), and in addition, may be stored at room temperature without risk of becoming contaminated. Glycerol is also commonly used in loading dyes but is not as versatile. Glycerol may not be used in TBE gels as the interaction with boric acid in the gel running buffer forms negatively charged esters that migrate in the gel and cause smearing of the nucleic acid bands. (J. Böeseken, Adv. Carbohydrate Chem., 4: 189-210 (1949).) It also requires refrigerated storage to prevent microbial contamination, as do sucrose containing loading dyes. A disadvantage of all the current formulas for nucleic acid loading compositions, including the use of Ficoll, is that they must be used in liquid form, thus requiring the use of a separate receptacle for mixing the nucleic acid and the loading composition together.
Another requirement for adding nucleic acids to the agarose gel well is the presence of a tracking dye to allow for visual assurance that the nucleic acid sample settled into the well and to monitor the progress of the nucleic acid as it travels through the gel to ascertain that it is moving in the correct direction. Loading dyes can be negatively charged, neutral, or positively charged. For example, U.S. Pat. No. 5,064,519 describes the use of neutral and positively charged dyes for nucleic acid electrophoresis. A neutral dye would simply stay in the well and not migrate with the nucleic acid, whereas a positively charged dye would run in the opposite orientation of the negatively charged nucleic acid. Commonly employed nucleic acid loading dyes contain slightly negatively charged dyes, such as bromophenol blue and xylene cyanol. These dyes provide the ability to visualize the migration of nucleic acids into the agarose and prevent over-run of nucleic acids.
In the conventional gel loading process, a liquid solution containing a tracking dye and a sedimenting agent is prepared and mixed with a liquid sample containing a macromolecule to be resolved by electrophoresis, e.g., a nucleic acid, a peptide, or a protein. Because the tracking dye and the sedimenting agent significantly increase the volume of the macromolecular sample, the operator must adjust the volume of the pipette before or after mixing to aspirate the entire sample before loading on a gel. When the number of samples is large, this additional step slows down the work flow and creates undesired delays.
Korean patent publication No. KR2000-0075140 by Kang et al. discloses the use of lyophilization (i.e. freeze-drying) to achieve a semi-dried or condensed version of a DNA molecular weight ladder using Ficoll™ or glycerol as the sugar. The problem with using glycerol for this purpose is that glycerol never reaches complete dryness, even with freeze-drying. This leads to an unstable format for transport as a product and produces variable volumes for gel loading. The semi-dried DNA molecular weight ladder disclosed by Kang et al. requires additional colored dye and EDTA for resuspension. In addition, the inventors discovered that the Ficoll™ dried molecular weight ladder appears distorted in TBE gels.
U.S. Pat. No. 5,861,251 discloses the use of lyophilized enzyme mixes containing dyes and sugar alcohols that are rehydrated upon contact with liquids. The dyes and sugar alcohols are provided for sedimentation and stabilization from heat damage. The stabilizer disclosed in U.S. Pat. No. 5,861,251 is glucitol (sorbitol), which the present inventors found to be unsuitable for use in the present invention because sorbitol does not have adequate density for holding nucleic acid in the agarose or acrylamide well under the buffer and cannot provide optimal resolution of nucleic acid bands in TBE gels.
U.S. Patent Pub. No. 2006/099567 discloses the use of trehalose as a sugar that provides stability to nucleic acids during drying. When the present inventors examined trehalose for its ability to be re-solubilized with the addition of nucleic acid in solution, they found that dried trehalose was very difficult to resuspend upon contact with liquid samples.
Accordingly, there is a need to develop a simple dry composition that contains all the components necessary for gel loading (i.e., a tracking dye and a sedimenting agent providing high density) and is easy to solubilize upon contact with a liquid sample. The present invention effectively addresses this need.